This invention relates to nucleic acids and encoded polypeptides which interact with the TGF-xcex2 superfamily receptor complexes and which are a negative regulators of TGF-xcex2 superfamily signalling. The invention also relates to agents which bind the nucleic acids or polypeptides. The invention further relates to methods of using such nucleic acids and polypeptides in the treatment and/or diagnosis of disease.
During mammalian embryogenesis and adult tissue homeostasis transforming growth factor xcex2 (TGF-xcex2) performs pivotal tasks in intercellular communication (Roberts et al., Growth Factors 8:1-9, 1993). The cellular effects of this pleiotropic factor are exerted by ligand-induced hetero-oligomerization of two distantly related type I and type II serine/threonine kinase receptors, Txcex2R-I and Txcex2R-II, respectively (Lin and Lodish, Trends Cell Biol. 11:972-978, 1993; Derynck, Trends Biochem. Sci. 19-:548-553, 1994; Massague and Weis-Garcia, Cancer Surv. 27:41-64, 1996; ten Dijke et al., Curr. Opin. Cell. Biol. 8:139-145, 1996). The two receptors, which both are required for signalling, act in sequence; Txcex2R-I is a substrate for the constitutively active Txcex2R-II kinase (Wrana et al., Nature 370:341-347, 1994; Weiser et al., EMBO J. 14:2199-2208, 1995).
TGF-xcex2 is the prototype of a large family of structurally related proteins that are involved in various biological activities (Massague, et al., Trends Cell Biol. 7:187-192, 1997; Roberts and Sporn, in: Peptide growth factors and their receptors, Part I (Sporn, M. B. and Roberts, A. B., eds) pp. 319-472, Springer-Verlag, Heidelberg (1990); Yingling et al., Biochim. Biophys. Acta 1242:115-136, 1995). The TGF-xcex2 xe2x80x9csuperfamilyxe2x80x9d includes activins and bone morphogenetic proteins (BMPs) that signal in a similar fashion, each employing distinct complexes of type I and type II serine/threonine kinase receptors (Lin and Lodish, 1993; Derynck, 1994; Massague and Weis-Garcia, 1996; ten Dijke et al., 1996). TGF-xcex2 related molecules act in environments where multiple signals interact and are likely to be under tight spatial and chronological regulation. For example, activin and BMP exert antagonistic effects in the development of Xenopus embryos (Graff et al., Cell 85:479-487, 1996). Chordin (Piccolo et al., Cell 86:589-598, 1996) and noggin (Zimmerman et al., Cell 86:599-606, 1996), for example, inhibit the ventralizing effect of BMP4 by binding specifically to the ligand. Likewise, follistatin neutralizes the activity of activin (Hemmati-Brivalou et al., Cell 77:283-295, 1994).
Genetic studies of TGF-xcex2-like signalling pathways in Drosophila and Caenorhabditis elegans have led to the identification of mothers against dpp (Mad) (Sekelsky et al., Genetics 139:1347-1358, 1995) and sma (Savage et al., Proc. Natl. Acad. Sci. USA 93:790-794, 1996) genes, respectively. The products of these related genes perform essential functions downstream of TGF-xcex2-like ligands acting via serine/threonine kinase receptors in these organisms (Wiersdorff et al., Development 122:2153:2163, 1996; Newfeld et al., Development 122:2099-2108, 1996; Hoodless et al., Cell 85:489-500, 1996). Vertebrate homologs of Mad and sma have been termed Smads (Derynck et al., Cell 87:173, 1996) or MADR genes (Wrana and Attisano, Trends Genet. 12:493-496, 1996). Genetic alterations in Smad2 and Smad4/DPC4 have been found in specific tumor subsets, and thus Smads may function as tumor suppressor genes (Hahn et al., Science 271:350-353, 1996; Riggins et al., Nature Genet. 13:347-349,1996; Eppert et al., Cell 86:543-552, 1996). Smad proteins share two regions of high similarity, termed MH1 and MH2 domains, connected with a variable proline-rich sequence (Massague, Cell 85:947-950, 1996; Derynck and Zhang, Curr. Biol. 6:1226-1229, 1996). The C-terminal part of Smad2, when fused to a heterologous DNA-binding domain, was found to have transcriptional activity (Liu et al., Nature 381:620-623, 1996; Meersseman et al., Mech. Dev. 61:127-1400, 1997). The intact Smad2 protein when fused to a DNA-binding domain, was latent, but transcriptional activity was unmasked after stimulation with ligand (Liu et al., 1996).
Different Smads specify different responses using functional assays in Xenopus. Whereas Smad1 induces ventral mesoderm, a BMP-like response, Smad2 induces dorsal mesoderm, an activin/TGF-xcex2-like response (Graff et al., Cell 85:479-487, 1996; Baker and Harland, Genes and Dev. 10:1880-1889, 1996; Thomsen, Development 122:2359-2366, 1996). Upon ligand stimulation Smads become phosphorylated on serine and threonine residues; BMP stimulates Smad1 phosphorylation, whereas TGF-xcex2 induces Smad2 and Smad3 phosphorylation (Hoodless et al., Cell 85:489-500, 1996; Liu et al., 1996; Eppert et al., 1996; Lechleider et al., J. Biol. Chem. 271:17617-17620, 1996; Yingling et al., Proc. Nat""l Aced. Sci. USA93:8940-8944, 1996; Zhang et al., Nature 383:168-172, 1996; Macias-Silva et al., Cell 87:1215-1224, 1996; Nakao et al., J. Biol. Chem. 272:2896-2900, 1996).
Smad4 is a common component of TGF-xcex2, activin and BMP signalling (Lagna et al., Nature 383:832-836, 1996; Zhang et al., Curr. Biol. 7:270-276, 1997; de Winter et al., Oncogene 14:1891-1900, 1997). Smad4 phosphorylation has thus far been reported only after activin stimulation of transfected cells (Lagna et al., 1996). After stimulation with TGF-xcex2 or activin Smad4 interacts with Smad2 or Smad3, and upon BMP challenge a heteromeric complex of Smad4 and Smad1 has been observed (Lagna et al., 1996). Upon ligand stimulation, Smad complexes translocate from the cytoplasm to the nucleus (Hoodless et al., 1996; Liu et al., 1996; Baker and Harland, 1996; Macias-Silva et al., 1996), where they, in combination with DNA-binding proteins, may regulate gene transcription (Chen et al., Nature 383:691-696, 1996).
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and agents which bind such polypeptides, including antibodies. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a Smad6 nucleic acid or polypeptide, or lack thereof. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, we present the identification of Smad6, which inhibits phosphorylation of pathway specific Smad polypeptides including Smad2 and Smad1 and inhibits the TGF-xcex2 superfamily signalling pathway such as the TGF-xcex2 and BMP signalling pathways.
According to one aspect of the invention, an isolated nucleic acid molecule is provided. The molecule hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ ID NO:1. The isolated nucleic acid molecule codes for a polypeptide which inhibits TGF-xcex2, activin, or BMP signalling. The invention further embraces nucleic acid molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code. The invention also embraces complements of the foregoing nucleic acids.
In certain embodiments, the isolated nucleic acid molecule comprises a molecule consisting of the nucleic acid sequence of SEQ ID NO:3 or consists essentially of the nucleic acid sequence of SEQ ID NO:1. Preferably, the isolated nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO:3,
According to another aspect of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule comprises a molecule consisting of a unique fragment of SEQ ID NO:3 between 12 and 1487 nucleotides in length and complements thereof, provided that the isolated nucleic acid molecule excludes sequences consisting only of SEQ ID NO:4. In one embodiment, the isolated nucleic acid molecule consists of between 12 and 32 contiguous nucleotides of SEQ ID NO:1, or complements of such nucleic acid molecules. In preferred embodiments, the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, or complements thereof.
According to another aspect of the invention, the invention involves expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to still other aspects of the invention, transgenic non-human animals are provided. The animals include in certain embodiments the foregoing expression vectors. In certain preferred embodiments, the transgenic non-human animal includes a conditional Smad6 expression vector, such as an expression vector that increases expression of Smad6 in a tissue specific, development stage specific, or inducible manner. In other embodiments, the transgenic non-human animal has reduced expression of Smad6 nucleic acid molecules. In some embodiments, the transgenic non-human animal includes a Smad6 gene disrupted by homologous recombination. The disruption can be homozygous or heterozygous. In other embodiments, the transgenic non-human animal includes a conditional Smad6 gene disruption, such as one mediated by e.g. tissue specific, development stage specific, or inducible, expression of a recombinase. In yet other embodiments, the transgenic non-human animal includes a trans-acting negative regulator of Smad6 expression, such as antisense Smad6 nucleic acid molecules, nucleic acid molecules which encode dominant negative Smad6 proteins, Smad6 directed ribozymes, etc.
According to another aspect of the invention, an isolated polypeptide is provided. The isolated polypeptide is encoded by the isolated nucleic acid molecule of any of claims 1, 2, 3, or 4, and the polypeptide has TGF-xcex2, activin, or BMP signalling inhibitory activity. Preferably, the isolated polypeptide consists of the amino acid sequence of SEQ ID NO:2.
In other embodiments, the isolated polypeptide consists of a fragment or variant of the foregoing which retains the activity of the foregoing.
According to another aspect of the invention, there are provided isolated polypeptides which selectively bind a Smad6 protein or fragment thereof, provided that the isolated polypeptide is not a TGF-xcex2 superfamily receptor, such as a TGF-xcex2, activin or BMP type I receptor. The isolated polypeptide in certain embodiments binds to a polypeptide encoded by the isolated nucleic acid molecule of any of claims 1, 2, 3, or 4. Preferred isolated polypeptides bind to an epitope defined by a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. In other preferred embodiments, isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to the Smad6 polypeptides of the invention). In still other preferred embodiments, the isolated polypeptide is a monoclonal antibody, a humanized antibody or a chimeric antibody.
The invention provides in another aspect an isolated complex of polypeptides. The isolated complex includes a TGF-xcex2 superfamily receptor or receptor complex bound to a polypeptide as claimed in claim 16. Preferably the isolated complex comprises a polypeptide having the amino acid sequence of SEQ ID NO:2. In other preferred embodiments, the receptor or receptor complex is selected from the group consisting of Txcex2RI, BMPR-IA, BMPR-IB, ActR-IA, a complex of Txcex2RI and Txcex2RII, a complex of BMPR-IA and BMPR-II, a complex of BMPR-IB and BMPR-II, a complex of ActR-IA and BMPR-II and a complex of ActR-IA and ActR-II.
According to still another aspect of the invention, methods for reducing TGF-xcex2 superfamily signal transduction in a mammalian cell are provided. The methods involve contacting a mammalian cell with an amount of an inhibitor of TGF-xcex2 superfamily signal transduction effective to reduce such signal transduction in the mammalian cell. Preferably the TGF-xcex2 superfamily signal transduction is mediated by a TGF-xcex2 superfamily ligand, particularly TGF-xcex21, activin, Vg1, BMP-4 and/or BMP-7. Other methods are provided for modulating phosphorylation of pathway specific Smads (e.g. Smad1, Smad2, Smad3 and/or Smad5). Certain methods are provided for reducing phosphorylation of Smad1 or Smad2 in a mammalian cell by contacting the mammalian cell with an agent which reduces Smad1 or Smad2 phosphorylation, respectively. Still other methods are provided for increasing phosphorylation of Smad3 in a mammalian cell by contacting the mammalian cell with an agent which increases Smad3 phosphorylation. In certain embodiments of the foregoing methods, the agent is an isolated Smad6 polypeptide, such as a polypeptide encoded by a nucleic acid which hybridizes under stringent conditions or the nucleic acid of SEQ ID NO:1, or degenerates or complements thereof.
According to still another aspect of the invention, methods for modulating proliferation and/or differentiation of a cancer cell are provided. The methods involve contacting a cancer cell with an amount of an isolated Smad6 polypeptide as described above, effective to reduce proliferation and/or differentiation of the cancer cell.
The invention in a further aspect provides methods for increasing TGF-xcex2 superfamily signal transduction in a mammalian cell. The mammalian cell is contacted with an agent that selectively binds to an isolated nucleic acid molecule of the invention or an expression product thereof in an amount effective to increase TGF-xcex2 superfamily signal transduction. Preferably the TGF-xcex2 superfamily signal transduction is mediated by a TGFxcex2 superfamily ligand selected from the group consisting of TGF-xcex21, activin, Vg1, BMP-4 and BMP-7. Preferred agents are antisense Smad6 nucleic acids, including modified nucleic acids, and polypeptides including antibodies which bind to a Smad6 polypeptide including the amino acids of SEQ ID NO:2, and a dominant negative variant of the polypeptide of SEQ ID NO:2.
The invention in still another aspect provides compositions comprising a Smad6 polypeptide and a pharmaceutically acceptable carrier.
The invention in a further aspect involves a method for decreasing Smad6 TGF-xcex2 superfamily inhibitory activity in a subject. An agent that selectively binds to an isolated nucleic acid molecule of the invention or an expression product thereof is administered to a subject in need of such treatment, in an amount effective to decrease TGFxcex2 superfamily signal transduction inhibitory activity of Smad7 in the subject. Preferably the TGFxcex2 superfamily signal transduction is mediated by a TGFxcex2 superfamily ligand selected from the group consisting of TGF-xcex2, activin, Vg1, BMP-4 and BMP-7. Preferred agents are antisense nucleic acids, including modified nucleic acids, and polypeptides including antibodies which bind to the polypeptide including the amino acids of SEQ ID NO:2, and dominant negative variants of the polypeptide of SEQ ID NO:2.
According to yet another aspect of the invention, methods for treating a condition characterized by abnormal BMP activity are provided. The methods include administering to a subject in need of such treatment an effective amount of Smad6 or a Smad6 agonist or antagonist sufficient to restore the BMP activity to normal. In some embodiments, the condition is selected from the group consisting of ossification of the posterior longitudinal ligament and ossification of the ligament flavum.
According to another aspect of the invention, methods for treating a condition characterized by abnormal TGF-xcex2 activity are provided. The methods include administering to a subject in need of such treatment an effective amount of Smad6 or a Smad6 agonist or antagonist sufficient to restore the TGF-xcex2 activity to normal. In certain embodiments, the condition is selected from the group consisting of liver fibrosis including cirrhosis and veno-occlusive disease; kidney fibrosis including glomerulonephritis, diabetic nephropathy, allograft rejection and HIV nephropathy; lung fibrosis including idiopathic fibrosis and autoimmune fibrosis; skin fibrosis including systemic sclerosis, keloids, hypertrophic burn scars and eosinophilia-myalgia syndrome; arterial fibrosis including vascular restenosis and atherosclerosis; central nervous system fibrosis including intraocular fibrosis; and other fibrotic diseases including rheumatoid arthritis and nasal polyposis.
In another aspect of the invention, methods for modulating the expression of cyclin A are provided. The methods include contacting a cell with Smad6 or an agonist or antagonist thereof, in an amount effective to modulate the expression of cyclin A. In some embodiments the cell is contacted with Smad6, and the expression of cyclin A is increased. In other embodiments, the cell is contacted with an antagonist of Smad6, and the expression of cyclin A is decreased. Preferably the antagonist of Smad6 is selected from the group consisting of antibodies to Smad6, dominant negative variants of Smad6 and Smad6 antisense nucleic acids.
According to another aspect of the invention, methods are provided for identifying lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated with Smad6 TGF-xcex2 superfamily signal transduction inhibitory activity. One set of methods involves forming a mixture of a Smad6 polypeptide, a TGF-xcex2 superfamily receptor or receptor complex, and a candidate pharmacological agent. The mixture is incubated under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of specific binding of the TGF-xcex2 superfamily receptor or receptor complex by the Smad6 polypeptide. A test amount of the specific binding of the TGF-xcex2 superfamily receptor or receptor complex by the Smad6 polypeptide then is detected. Detection of an increase in the foregoing activity in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases the Smad6 TGF-xcex2 superfamily signal transduction inhibitory activity. Detection of a decrease in the foregoing activities in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which decreases Smad6 TGF-xcex2 superfamily signal transduction inhibitory activity. Another set of methods involves forming a mixture as above, adding further a pathway specific Smad polypeptide, and detecting first and test amounts of TGF-xcex2 superfamily induced phosphorylation of the pathway specific Smad polypeptide. Detection of an increase in the phosphorylation in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which decreases the Smad6 TGF-xcex2 superfamily signal transduction inhibitory activity. Detection of a decrease in the foregoing activities in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases Smad6 TGF-xcex2 superfamily signal transduction inhibitory activity. Preferred Smad6 polypeptides include the polypeptides of claim 16. Preferably the TGFxcex2 superfamily receptor is selected from the group consisting of TGFxcex2 superfamily type I receptors, TGFxcex2 superfamily type II receptors, and complexes of TGFxcex2 superfamily type I receptors and TGFxcex2 superfamily type II receptors. Preferred pathway specific Smad polypeptides include Smad1 and Smad2.
According to still another aspect of the invention, methods for increasing phosphorylation of Smad3 in a mammalian cell are provided. The methods include contacting the mammalian cell with an amount of an isolated Smad6 polypeptide effective to increase phosphorylation of Smad3 in the mammalian cell.
According to another aspect of the invention, methods for reducing heteromerization of Smad2 with Smad3 or Smad4 in a mammalian cell are provided. The methods include contacting the mammalian cell with an amount of an isolated Smad6 nucleic acid or polypeptide, or an agonist thereof, effective to reduce heteromerization of Smad2 with Smad3 or Smad4 in the mammalian cell.
The use of the foregoing compositions, nucleic acids and polypeptides in the preparation of medicaments also is provided.
In the foregoing compositions and methods, preferred members of the TGF-xcex2 superfamily are TGF-xcex21, activin, Vg1, BMP-4 and BMP-7, and the preferred pathway specific Smad polypeptides are Smad1, Smad2, Smad3 and Smad5.
These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.